Professor Dr. Richard Bruynoghe: A 1951 overview of his bacteriophage research spanning three decades

In 1921, Richard Bruynoghe and his student Joséph Maisin published on the first use of bacteriophages in a phage therapy context. At that time, Bruynoghe (a medical doctor) was affiliated as a professor at the KU Leuven (Belgium) for just over a decade, within the Bacteriological Institute which he founded and led. After a distinguished career (he was acting mayor of the city of Leuven-Belgium during the second World War), he received a special medical award in 1951 just before his retirement in 1952. In this perspective, he was asked to provide an overview of his research for a lay-audience within the local University magazine: Onze Alma Mater (Our alma mater). We, as current affiliates of the KU Leuven are honored to present some of his legacy, which to date has been largely overlooked in historical accounts

Synthetic biology of modular enzymes: From enzymes to enzybiotics

Over the past few years antimicrobial resistance has evolved from a rare event to an everyday occurring problem in health care. The future looks even more grim due to the increase of antimicrobial resistance against antibiotics and the unprecedented discovery void of new antibiotic classes. Enzyme-based antimicrobials or enzybiotics represent a novel class of antibacterials. Specifically, endolysins encoded by bacterial viruses (bacteriophages) that degrade the peptidoglycan layer, have gained tremendous interest with many proof-of-concept studies up to clinical phase studies. Initially, native endolysins were only considered for Gram-positive bacteria as the peptidoglycan layer of Gram-negative bacteria is protected by the outer membrane. However, Gram-negative pathogens constitute the largest threat for health care given the higher extent of multi- and pan-drug resistance and lower number of recently developed antibiotics or antibiotics in the pipeline. Using enzyme engineering, we have expanded the spectrum to Gram-negative bacteria. This is achieved by fusing outer membrane permeabilizing peptides via a linker to endolysins. The peptide locally destabilizes the outer membrane and transfers the endolysin moiety across the outer membrane, followed by active peptidoglycan degradation. Exposure of Gram-negative bacteria to these engineered enzybiotics (Artilysin®s) results in a prompt, highly bactericidal effect, which has been confirmed in in vitro keratinocyte cultures and nematodes. Case studies in wound care treatment of dogs have shown a successful outcome. Enzymes are an unusual source for the development of antibacterials, but we have shown that exactly this enzymatic nature provides these engineered enzybiotics with novel features for antibacterials. First, they are rapid and act immediately upon contact. Real-time time-lapse microscopy shows that cells are killed within seconds. Second, they show no cross-resistance with existing antibiotics due to the novel mode-of-action and do not provoke resistance development. Third, they actively degrade all bacterial cells regardless if they are metabolically active or not, whereas traditional antibiotics require an active metabolism. Therefore, engineered enzybiotics are able to kill metabolically dormant persisters that cause recurrent, chronic infections (Briers et al., 2014; Gerstmans et al., 2016). A unique feature of this class of enzybiotics is the engineering potential. They are modular proteins, comprising different domains: an outer membrane permeabilizing peptide, a linker sequence and an endolysin, which in turn comprises a cell wall binding domain and an enzymatically active domain. Depending on the modular composition and the specific order of modules, its enzymatic and antibacterial properties, expression level and stability can be modulated. Using combinatorial shuffling in a synthetic biology approach, we show how targeted antibacterials with diverse properties can be constructed. In sum, engineered enzybiotics provide a platform approach for customized development of antibacterials with unique features based on their enzymatic nature. Briers, Y., Walmagh, M., Van Puyenbroeck, V., Cornelissen, A. Cenens, W., Aertsen, A., Oliveira, H., Azeredo, J., Verween, G., Pirnay, J.-P, Volckaert, G., and Lavigne, R. (2014a) Engineered endolysin-based ‘Artilysins’ to combat multidrug-resistant Gram-negative pathogens. MBio 5:e01379-14. Gerstmans, H., Rodriguez-Rubio, L., Lavigne, R., Briers, Y. (2016) From endolysins to Artilysins: novel enzyme-based approaches to kill drug-resistant bacteria. Biochem Soc T 44:123-12

Phage-based target discovery and its exploitation towards novel antibacterial molecules

The deeply intertwined evolutionary history between bacteriophages and bacteria has endowed phages with highly specific mechanisms to hijack bacterial cell metabolism for their propagation. Here, we present a comprehensive, phage-driven strategy to reveal novel antibacterial targets by the exploitation of phage-bacteria interactions. This strategy will enable the design of small molecules, which mimic the inhibitory phage proteins, and allow the subsequent hit-to-lead development of these antimicrobial compounds. This proposed small molecule approach is distinct from phage therapy and phage enzyme-based antimicrobials and may produce a more sustainable generation of new antibiotics that exploit novel bacterial targets and act in a pathogen-specific manner.Peer reviewe

The SPO1-related bacteriophages

A large and diverse group of bacteriophages has been termed ‘SPO1-like viruses'. To date, molecular data and genome sequences are available for Bacillus phage SPO1 and eight related phages infecting members of other bacterial genera. Many additional bacteriophages have been described as SPO1-related, but very few data are available for most of them. We present an overview of putative ‘SPO1-like viruses' and shall discuss the available data in view of the recently proposed expansion of this group of bacteriophages to the tentative subfamily Spounavirinae. Characteristics of SPO1-related phages include (a) the host organisms are Firmicutes; (b) members are strictly virulent myoviruses; (c) all phages feature common morphological properties; (d) the phage genome consists of a terminally redundant, non-permuted dsDNA molecule of 127-157kb in size; and (e) phages share considerable amino acid homology. The number of phages isolated consistent with these parameters is large, suggesting a ubiquitous nature of this group of viruse

SHORT COMMUNICATION - Functional complementation in Saccharomyces cerevisiae under control of the natural yeast promoter

A novel approach for functional complementation of foreign genes in Saccharomyces cerevisiae is presented. This approach is based on the use of the widely available cognate gene plasmids (e.g. pRS416) of the European Functional Analysis Network (EUROFAN). The functional complementation of the human homolog of YOR159c (SME1 gene) shown here is the first demonstration of complementation using the original yeast promoter, theoretically offering a more natural regulation of protein expression

Bacteriophage endolysins as a response to emerging foodborne pathogens

Continuous reports on foodborne outbreaks and increasing prevalence of antibiotic-resistant bacteria call for the development of novel preservation techniques that assure the safety of food products. Bacteriophage endolysins are highly active antibacterial peptidoglycan hydrolases that have evolved over millions of years to become the ultimate weapon against bacteria, with potential to be used as a food preservative. We here give an overview of all distinct endolysins encountered so far, we discuss their inherent qualities and review their role in preventing and controlling foodborne pathogens, divulging their potential for future applications.This work was supported by a grant from the Portuguese Foundation for Science and Technology in the scope of the Projects PTDC/AGR-ALI/100492/2008 and PTDC/AGR-ALI/121057/2010. Hugo Oliveira is paid by the FCT grant SFRH/BD/63734/2009

Pro- and anti-inflammatory responses of peripheral blood mononuclear cells induced by Staphylococcus aureus and Pseudomonas aeruginosa phages

The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes (i.e. CD14, CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, IL10, LYZ, SOCS3, TGFBI and TNFA) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages (i.e. PNM, LUZ19, 14- 1 and GE-vB_Pae-Kakheti25). The phages were able to induce clear and reproducible immune responses. Moreover, the overall immune response was very comparable for all five phages: down-regulation of LYZ and TGFBI, and up-regulation of CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, SOCS3 and TNFA. The observed immune response was shown to be endotoxinin-dependent and predominantly anti-inflammatory. Addition of endotoxins to the highly purified phages did not cause an immune response comparable to the one induced by the (endotoxin containing) phage lysate. In addition, the use of an intermediate level of endotoxins tipped the immune response to a more anti-inflammatory response, i.e. up-regulation of IL1RN and a strongly reduced expression of CXCL1 and CXCL5

Experimental phage therapy of burn wound infection : difficult first steps

Antibiotic resistance has become a major public health problem and the antibiotics pipeline is running dry. Bacteriophages (phages) may offer an ‘innovative’ means of infection treatment, which can be combined or alternated with antibiotic therapy and may enhance our abilities to treat bacterial infections successfully. Today, in the Queen Astrid Military Hospital, phage therapy is increasingly considered as part of a salvage therapy for patients in therapeutic dead end, particularly those with multidrug resistant infections. We describe the application of a well-defined and quality controlled phage cocktail, active against Pseudomonas aeruginosa and Staphylococcus aureus, on colonized burn wounds within a modest clinical trial (nine patients, 10 applications), which was approved by a leading Belgian Medical Ethical Committee. No adverse events, clinical abnormalities or changes in laboratory test results that could be related to the application of phages were observed. Unfortunately, this very prudent ‘clinical trial’ did not allow for an adequate evaluation of the efficacy of the phage cocktail. Nevertheless, this first ‘baby step’ revealed several pitfalls and lessons for future experimental phage therapy and helped overcome the psychological hurdles that existed to the use of viruses in the treatment of patients in our burn unit

Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing

Protein engineering and synthetic biology applications increasingly rely on the assembly of modular libraries composed of thousands of different combinations of DNA building blocks. At present, the validation of such libraries is performed by Sanger sequencing analysis on a small subset of clones on an ad hoc basis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creation in vitro, using long reads sequencing technology. After an initial step of nanopore sequencing, we use straightforward bioinformatics tools to tabulate the composition and synteny of the building blocks in each read. We subsequently use exploratory statistics to assess the library and validate its diversity before carrying downstream cloning and screening assays